Intein-mediated backbone cyclization of entolimod confers enhanced radioprotective activity in mouse models

Background. Entolimod is a Salmonella enterica flagellin derivate. Previous work has demonstrated that entolimod effectively protects mice and non-human primates from ionizing radiation. However, it caused a “flu-like” syndrome after radioprotective and anticancer clinical application, indicating some type of immunogenicity and toxicity. Cyclization is commonly used to improve the in vivo stability and activity of peptides and proteins. Methods. We designed and constructed cyclic entolimod using split Npu DnaE intein with almost 100% cyclization efficiency. We adopted different strategies to purify the linear and circular entolimod due to their different topologies. Results. After Nichelating affinity purification, the linear and circular entolimod were purified by sizeexclusion and ion-exchange chromatography, respectively. Compared with linear entolimod, the circular entolimod showed significantly increased both the in vitro NF-κB signaling and in vivo radioprotective activity in mice. Discussions/Conclusions. Our data indicates that circular entolimod might be a good candidate for further clinical investigation.